KR20100106599A - Attenuated recombinant alphaviruses incapable of replicating in mosquitoes and uses thereof - Google Patents

Abstract

The present invention discloses attenuated recombinant alphaviruses that are not replicable in mosquito cells and that are not infectable by mosquito vectors. These attenuated alphaviruses include Western Equine Encephalitis Virus, Eastern Equine Encephalitis Virus, Venezuelan Equine Encephalitis virus, and Chikunungunya virus. However, this is not limited. The present invention also discloses such alphaviruses and their use in immunogenic compositions.

Description

Attenuated Recombinant Alphaviruses Incapable of Replicating in Mosquitoes and Uses Thereof}

The present invention relates to the field of molecular biology, virology and immunology of alphaviruses. More specifically, the present invention provides attenuated recombinant alphaviruses having a mosquito inability to infect, and reveals the methods of producing such alphaviruses and the use of these attenuated alphaviruses in immunogenic compositions.

Alphavirus genes in the Togaviridae family include a number of important human and animal pathogens. These viruses are widely distributed on all continents except Antarctica and represent important public health threats (18, 39). Under natural conditions, most alphaviruses are transmitted by mosquitoes, where they cause persistent long-lived infections that have little effect on the biological function of the vector. In vertebrates infected by mosquitoes during their blood intake, alpha viruses cause acute infections characterized by high-titer virus infection and its natural circulation, which is a prerequisite for new mosquito infections.

Venezuelan equine encephalitis virus (VEEV) is one of the most potent pathogenic members of the alphavirus gene. It circulates continuously in southern, central and northern America, causing sporadic epidemics and epizootics associated with humans, horses and other livestock. During the most recent major outbreaks in Venezuela and Colombia (1995) in connection with subtype IC VEEV, it occurred in about 100,000 people and more than 300 fatal encephalitis cases were investigated (37). During VEEV livestock epidemic, horse death due to encephalitis can reach 83%, while the overall mortality rate is low in humans (<1%), while disorientation, ataxia, and mental depression Mental disorders can be detected up to 14% of infected individuals, especially in children (21). Human diseases caused by VEEV are characterized by fever with chills, severe headache, myalgia, somnolence, and pharyngitis. Young and old individuals develop into reticuloendothelial infections with severe lymph depletion followed by encephalitis. The result of CNS infection is acute meningoencephalitis, which causes neuronal cells to die (9). Neural signals appear within 4 to 10 days of the onset of the disease and include seizures, mild paresis, behavioral changes, and coma.

Despite the continuing threat of the VEEV pandemic, no safe and effective vaccine against this virus has been designed. The attenuated TC-83 strain of VEEV was developed more than 40 years ago by a series of passages of the highly venomous Trinidad donkey (TRD) strain of VEEV in guinea pig heart cells ( 4). Finally, TC-83 is still the only vaccine available for lab workers and soldiers. More than 8,000 people were vaccinated (2,4,34), and cumulative data clearly indicate that nearly 40% of all vaccines develop into diseases with some of the typical symptoms of native VEEV, including fever, systematic disease, and other adverse effects. (2). This TC-83 strain commonly kills non-adult mice, but not adult mice, after cerebral (ic) and subcutaneous (sc) inoculation (31), and therefore is excellent for the study of further attenuation and effects of mutations in viral outbreaks. Starting material.

The VEEV genome is similar to the structure of cellular mRNA and is a positively polar single-stranded RNA molecule, nearly 12-kb-long. The genomic RNA comprises both a 5 'methylguanylate cap and a 3' polyadenylate tail (24) and is characterized by allowing translation of viral proteins by host cell tissue immediately after release of genomic RNA from nucleocapsids. It includes. 5'-2 / 3 of the genome are translated into nonstructural proteins (nsPs) containing the viral components of the replicative enzyme complex required for replication of the viral genome and transcription of subgenomic RNA. Subgenomic RNA corresponds to the 3 'third of the genome. It is synthesized from the subgenomic promoter and translated into viral structural proteins. The attenuated phenotype of VEEV strain TC-83 is the result of two mutations in the strain TRD genome, one of which replaced the amino acid at position 120 of the E2 glycoprotein, and the second at nt 3 at 5'UTR. Change (11, 23, 24, 43). Thus, due to the very high mutation rate of alphaviruses, the reversion from TC-83 to the pathogenic phenotype is a major problem when appropriate selective conditions such as viral passage in vivo occur. In addition, VEEV TC-83 is a replicating and mosquito-infected in mosquito cells that can be vaccinated (32), thus still delivering by mosquitoes.

Ideally, live arbovirus vaccine strains should not be delivered by arthropod vectors because circulation in the host can lead to unpredictable changes, including increasing toxicity. In particular attenuated strains generated from wild-type viruses that rely on a few attenuated mutations where conversion can occur, or genetic modifications that may evolve in unexpected ways if they have undergone a vector-borne circulation This is even more so for the strains that have been identified. The former risk was emphasized by the detection of the VEEV TC-83 vaccine strain in mosquitoes collected outside of the pandemic / pandemic restricted to Louisiana, Texas in 1971 (32).

The development of infectious cDNA against alphaviruses opens the opportunity to analyze their weakness by making extensive modifications to the viral genome, an approach that can minimize or exclude conversion to the wild type (wt) pathogenic phenotype. In addition, the genome of these alphaviruses can be engineered to contain RNA elements that function only in cells of vertebrate origin, not insects. Thus, such a wide range of mutations can prevent the transmission of genetically modified viruses by mosquito vectors.

Despite the importance of human and animal pathogenicity, potential as biological weapons and the application of attenuated alphaviruses, the prior art has shown that methods for producing attenuated strains of alphaviruses that can only replicate in vertebrate cells are available. Lack. The present invention meets the needs and desires for a long time in the prior art.

One embodiment of the present invention provides a method for generating an attenuated recombinant alphavirus. This method uses an encephalomyelocarditis virus (EMCV IRES) between the end of the nonstructural protein 4 (nsP4) coding sequence instead of the native 5 'UTR of the subgenomic RNA and the starting AUG of the subgenomic RNA coding sequence of the alphavirus. Cloning the internal ribosomal entry site. In another related embodiment of the invention, an attenuated recombinant alphavirus produced by the method discussed above is provided.

In another related embodiment of the invention, a vector comprising a nucleotide sequence encoding an attenuated recombinant alphavirus and a host cell comprising and expressing the vector are provided. In another related embodiment of the invention, a pharmaceutical composition is provided. The composition comprises the attenuated recombinant alphavirus discussed above and a pharmaceutically acceptable carrier. In a related embodiment of the invention, an immune composition is provided. The immune composition comprises the attenuated recombinant alphavirus described above.

In another related embodiment of the invention, a method of protecting a subject from infection following exposure to an alphavirus is provided. Such methods include administering an immunologically effective amount of an immunogenic composition comprising the attenuated recombinant alphavirus described herein to protect the subject from infection following exposure to the alphavirus.

The present invention is directed to new strategies for developing attenuated strains of alphaviruses. These attenuated alphaviruses can only replicate in vertebrate cells. This phenotype is obtained by providing translation of viral structural proteins and viral replication that ultimately depends on the internal ribosomal entry site of encephalomyelocarditis virus (EMCV IRES). This recombinant virus is viable and shows a highly attenuated phenotype, induced protective immunity against VEEV infection in newly born mice. In addition, formalin-inactivated vaccines are expensive and ineffective. In addition, these vaccines also require multiple repeated vaccinations. In addition, the only useful live attenuated vaccine against VEEV infection infects mosquitoes during blood intake in reactogenic vaccinated horses. The attenuated alphaviruses discussed herein offer significant advantages over currently available vaccines because the attenuated phenotype is irreversible. In addition, genetically engineered alphaviruses are unable to replicate in mosquito cells and, as they cannot replicate in mosquitoes, suggest new approaches for generating new live recombinant viruses that cannot be circulated in nature.

Development of infected cDNA clones from Sindbis and other alphaviruses (12, 25, 36) is a novel recombinant vaccine as well as for reverse genetics experiments, which are the research goals in other aspects of viral biology and pathology. Opened up opportunities for development. Attenuation of the virus by tissue culture or passage through chicken embryos (29) generally results in few point mutations in structural and nonstructural genes of the viral genome and in cis-acting elements. Is obtained from the accumulation of. For example, the VEEV TC-83 vaccine strain relies solely on two-point mutations for its attenuation, and a high degree of reactivity (34) probably reflects the instability of this attenuation mechanism. This raises the issue of possible conversion to the wild type (wt) pathogenic phenotype during viral replication in vaccinated individuals. The number of mutations can be further increased by chemical mutagenesis (28), but this process also does not irreversibly introduce the introduced changes. Infection of RNA ⁺ Virus Genetic manipulation with cDNA clones opens up great potential for more powerful modifications of the viral genome, making extensive deletions impossible to convert to wild type (wt) genomic sequence (5,6,10,19). Or to provide additional genetic material that enhances the immunogenicity of the variant.

Genetically altered arboviruses are also introduced into the natural circulation mediated by mosquito vectors, and there is also a big problem of showing further evolution during long term replication in mosquitoes or during the development of viremia in vertebrate hosts. One example is the use of VEEV TC-83, which can produce levels of Vilemia in horses sufficient to infect mosquitoes. Isolation of TC-83 from naturally infected mosquitoes collected in Louisiana (32) during the 1971 Texas epidemic highlighted the risk of attenuated alphavirus transmission. Thus, in designing new generations of live vaccine strains, it is prudent to make highly attenuated arboviruses as well as replicate only in the cells of vertebrates. This can be accomplished by cloning cell-specific RNA elements into the viral genome. In contrast to the cricket paralysis virus IRES (20), EMCV-specific elements were expected to function very poorly in arthropod cells.

In the present invention, EMCV IRES was cloned into the VEEV TC-83 genome for translation of the viral structural gene IRES-dependent. One of the genomes contained a functional subgenomic promoter and included an IRES at the 5'UTR of the subgenomic RNA. Although the virus is alive, its ability to produce subgenomic RNA promotes further evolution, which resulted in IRES deletions and conversions to standard, cap-dependent translation of structural proteins. This latter deletion allows it to replicate in mosquito cells. The second variant from the subgenomic promoter to multiple mutations is stable in terms of its inability to switch to cap-dependent translation. Since this conversion requires IRES deletions as well as the recovery of subgenomic promoters inactivated by 13 mutations, direct reversion of these multiple mutations presents a potentially negligible risk. However, this variant has developed in an interesting way to evolve into a phenotype that replicates more effectively by accumulating additional, adaptive mutations in the nsP2 gene. These mutations do not noticeably change the level of viral RNA replication, synthesis of viral structural proteins, or their compartmentalization in the cell. The detected mutations also do not produce additional signals that can increase the efficacy of genomic packaging. Thus, the mechanism of these functionalities remains to be determined. However, it was speculated that the accumulation of published data suggests that genomic packaging of RNA ⁺ viruses is largely determined by replication complexes, and that the genome needs to be present as functional nsPs in structural proteins for particle formation (22, 30). The working hypothesis in the present invention is that the helicase domain of nsP2 can be associated with the presentation of the viral genome for its packaging with nucleocapsids, and thus the identified mutations are It has a positive effect on the efficiency of the process.

It should be clarified that the object of the present invention is to develop VEEV variants that exhibit a non-replicable, stable and more attenuated phenotype in arthropod vectors. Slow growth of VEEV / mutSG / IRES / 1 variants designed in both IFN-alpha / beta-deficient and IFN signaling-deficient BHK-21 cells, their ability to induce high levels of IFN-alpha / beta in tissue culture, cerebral (ic) Their greatly reduced ability to kill newly born mice even after inoculation, and their non-replicability in mosquito cells, suggest that these variants meet this requirement. Its immunity will be further investigated in other animal models. In addition, other encephalogenic alphaviruses can be attenuated using similar EMCV IRES-based strategies, which can be applied in combination with other recently developed approaches (1, 13-15, 31).

In summary, the object of the present invention is the development of attenuated alphaviruses and other alphaviruses such as encephalitis alphaviruses, including VEEV, EEEV and WEEV, and Chikkununya virus, which cause disease in humans and livestock. To a new type of vaccine for their application. Replication of these alphaviruses is dependent on EMCV IRES, which makes their replication impossible in mosquito cells or mosquito vectors. More importantly, this phenotype is irreversible because extensive modifications are introduced into the viral genome. Thus, these new variants can be used for vaccination without problems with their potential for transmission by natural mosquito vectors.

The present invention relates to a method for generating an attenuated recombinant alphavirus, said method comprising an encephal between the end of a nonstructural protein (nsP4) coding sequence instead of the native 5 'UTR and the starting AUG of the subgenomic RNA coding sequence of the alphavirus. Cloning the internal ribosomal entry site of Myelocarditis virus (EMCV IRES). The method further comprises inactivating the subgenomic promoter of the alphavirus by clustered point mutations and deletion of the native 5 ′ UTR in the subgenomic RNA. In addition, inactivation of the subgenomic promoter will not modify the carboxy terminal of nonstructural protein 4. Additionally, the method may further comprise introducing an adaptive mutation in nonstructural protein 2 (nsP2) that is effective for increasing viral replication, release and viral titer. Examples of adaptive mutations in nonstructural protein 2 include, but are not limited to, Y ₃₇₀ → C, K ₃₇₁ → Q, P ₃₄₉ → T, D ₄₁ 8 → A, K ₄₂₃ -T, or a combination thereof. . Also, examples of alphaviruses include, but are not limited to, Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Western Equine Encephalitis Virus or Chikunguniya Virus (WEEV).

The present invention relates to attenuated recombinant alphaviruses produced by the methods discussed above. These alphaviruses are unable to replicate in mosquitoes, cannot be transmitted by mosquito vectors, induce high levels of IFN alpha / beta, slow growth in IFN alpha / beta cells, slow growth in IFN signaling-deficient BHK-21 cells Or a combination thereof may be possible.

The invention also relates to a vector comprising a nucleotide sequence encoding the attenuated recombinant alphavirus discussed above and a host cell comprising and expressing the vector. Constructing vectors and expressing them in cells is well known and is a standard technique in the art. Thus, those skilled in the art can build such vectors based on the common experiments and knowledge available in the art.

The invention also relates to a pharmaceutical composition comprising the attenuated recombinant alphavirus discussed above and a pharmaceutically acceptable carrier. The invention also relates to an immunogenic composition comprising the attenuated recombinant alphavirus herein.

The invention also provides a method of protecting a subject from infection following exposure to alphaviruses, comprising administering an immunologically effective amount of the immunogenic composition discussed above to protect the subject from infection following exposure to alphaviruses. It is about. The subjects benefiting from the above methods may be humans or livestock.

The present invention also includes cloning the internal ribosomal entry site of Encephalomyelocarditis virus and the capsid gene downstream of the envelope glycoprotein gene at the 3 'end of the upstream immediately before the subgenomic region of 3'UTR with the capsid gene. Wherein the capsid is expressed in a cap-independent manner, except that the capsid protein gene is translated in an IRES-dependent manner, and the envelope protein gene is translated in a cap-dependent manner. The present invention relates to a method for producing an alphavirus.

As used herein, the indefinite article ("a") or ("an") means one or more. As used in the claims, when used in combination with the word "comprising", the indefinite article "a" or "an" means one or more than one. As used herein, "another" or "other" means at least two or more of the same or different elements of the claims or components thereof.

The compositions described herein are systematically or topically by any standard method in the art, for example, subcutaneously, intravenously, parenterally, intraperitoneally, skin, intramuscularly, locally, or nasal. It can be administered independently. Dosage formulations of the compositions described herein are suitable for the method of administration, and may include conventionally non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles well known to those skilled in the art.

The compositions described herein can be administered independently one or more times to achieve, maintain or ameliorate a therapeutic effect. It is well known to those skilled in the art to determine the dosage or whether the proper administration of the composition is a single dose or multiple doses. Suitable dosages will depend on the health of the subject, the induction of immune responses and / or prophylaxis by alphaviruses, the mode of administration and the formulation used.

According to the present invention, there is provided an attenuated recombinant alpha virus which is impossible to replicate in mosquito cells and is not transmitted by a mosquito vector, and by using the same, prevention of western equine encephalitis virus, eastern equine encephalitis virus, chikuguniya virus, etc. It can be usefully used as an immunogenic composition for.

1A-1D show EMCV IRES-encoding and replication of VEET TC-83-derived recombinant virus in BHK-21 cells. 1A shows designed viral genome, infectivity of in vitro-synthesized RNA in infection center assay, virus titer in late 24-hour transfection of 1 mg of in vitro-synthesized RNA into BHK-21 cells, and 48 The size of the plaque formed by viruses in BHK-21 cells in late transfection is shown schematically. Arrows indicate functional subgenomic promoters. The filled box represents the EMCV IRES. 1B shows the alignment of subgenomic promoter-containing fragments of the VEEV TC-83 genome and corresponding fragments of VEEV / mutSG / IRES. The position of the promoter is indicated by an empty box. The start of subgenomic RNA and the start of EMCV IRES in the VEEV TC-83 genome are indicated by arrows. Mutations introduced into the VEEV / mutSG / IRES genome are shown in lowercase. 1C shows plaques formed in BHK-21 cells by virus harvested in late 24 hours transfection. 1D shows replication of the virus after transfection of 1 mg of in vitro-synthesized RNA with BHK-21 cells.
2A-2C show mutations found in plaque-purified VEEV / mutSG / IRES variants, which are more effective replication in BHK-21 cells and defined adaptations in VEEV TC-83 and VEEV / mutSG / IRES replication. ) Shows the effect of the mutation. 2A shows a list of mutations found in the genome of plaque isolates compared to the published sequence of VEEV TC-83 (24). FIG. 2B is a schematic diagram of the VEEV TC-83 and VEEV / mutSG / IRES genomes with infectivity of in vitro-synthesized viral RNA in one or both identified mutations and infection center analysis. Functional subgenomic promoters are indicated by arrows and EMCV IRES are indicated by filled boxes. 2C shows replication of the designed virus in BHK-21 cells after transfection of 1 mg of in vitro-synthesized viral genome.
3A-3B show mutations identified in the nsP2 protein of the VEEV / mutSG / IRES variant showing the large-flag phenotype. FIG. 3A additionally three times in the genome of plaque-purified isolates from virus stocks harvested in late 24-hour transfection of in vitro-synthesized RNA (Orig.) And in Vero cells (Pass.) In the genome of isolates from passaged stocks, a list of identified mutations is shown. 3B shows the location of the mutations defined in VEEV nsP2. The location of the currently known functional domains in alphavirus nsP2 (38, 39) is shown.
4A-4B show analysis of protein and RNA synthesis in BHK-21 cells transfected with in vitro-synthesized recombinant viral RNA. The cells were electroporated with 4 mg of the indicated RNA and placed in a 35-mm dish. In FIG. 4A, in late 4.5 hours transfection, the medium in the wells was replaced with 1 ml of aMEM with 10% FBS, ActD (1 mg / ml) and [ ³ H] uridine (20 mCi / ml) added. It was. After 4 hours of incubation at 37 ° C, RNA was isolated and analyzed by agarose gel electrophoresis. The location of the viral genome and subgenomic RNA is shown by G and SG, respectively. VEEV / IRES-specific subgenomic RNA forms a more diffused band than does other subgenomic RNA-producing viruses, because in the gel it migrated together to ribosomal 28S RNA. In FIG. 4B, at late 12 h transfection, the protein was metabolically labeled with [ ³⁵ S] methionine and analyzed on sodium dodecyl sulfate-10% polyacrylamide gel. The location of the molecular weight marker (kDa) is shown on the left side of the gel. Location of viral structural proteins: C, El and p62 (precursors of E2) are shown on the right side of the gel. Asterisks indicate the location of cellular proteins (heat-shock proteins) induced by replication of the IRES-encoding virus.
5A-5C show the passaging of recombinant EMCV IRES-containing VEEV variants in C ₇ IO cells. 5A is a schematic diagram of the viral genome. The arrow shows the position of the functional subgenomic promoter. The filled boxes indicate the location of the EMCV IRES. 5b shows the titer of recombinant virus after passaging in C ₇ 10 cells. Cells in 35-mm dishes were infected with 40 ml of virus sample harvested in late 24-hour transfection of BHK-21 cells or in late 48-hour infection of C ₇ 10 cells with in vitro-synthesized RNA (P1). The dashed line indicates the limit of detection. 5C shows the deletion of IRES-containing sequences identified in plaque-purified VEEV / IRES variants showing effective replication in C ₇ 10 cells. The remaining EMCV IRES-specific sequences are shown in lowercase letters.
6 shows replication of VEEV / mutSG / IRES / 1 and VEEV TC-83 in NIH 3T3 cells. Cells were infected at MOI of 10 PFU / cell. The medium was replaced at the indicated time points and virus titers measured by plaque analysis in BHK-21 cells. The same samples were used to measure IFN-a / b release in biological assays. The concentration of IFN-a / b released is expressed in international units (IU) per ml.
7 shows survival of mice infected with VEEV TC-83 and VEEV / mutSG / IRES / 1 virus. Six-day-old NIH Swiss mice were cerebral (ic) inoculated with approximately 10 ⁶ PEU of the indicated virus. Animals were monitored for 2 months. None of these experiments had deaths occurring after 9 days of post-infection.
8 shows survival following vaccination and challenge of adult mice. Female NIH Swiss mice 5-6 weeks old were subcutaneously (sc) immunized with a VEEV strain TC-83 or recombinant virus at a dose of about 10 ⁶ PFU. Three weeks post immunization, mice were challenged subcutaneously (sc) with approximately 10 ⁴ PFU of VEEV strain 3908 and deaths were recorded.
9 shows a schematic method of generating attenuated recombinant alphaviruses that are incapable of mosquito infection.

The following examples are provided for the purpose of illustrating various embodiments of the invention and do not imply that the invention is limited in this manner. Those skilled in the art will readily appreciate that the present invention is suitable for carrying out the above objects and achieving the stated results and advantages, as well as these objects. Changes within this and other uses that fall within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example  1: cell culture

BHK-21 cells were provided by Paul Olivo (Washington University, St. Louis, Mo) and Vero cells were provided by Charles Rice (Rockefeller University, NY, NY). NH 3T3 cells were obtained from the American Type Tissue Culture Collection (Manassas, VA). These cell lines were maintained at 37 ° C. in alpha minimal essential nutrient medium (aMEM) supplemented with 10% fetal calf serum (FBS) and vitamins. Mosquito C ₇ 10 cells were obtained from Henry Huang (Washington Univ., St. Louis, Mo.) and 10% heat-inactivated FBS and 10% tryptose phosphate broth (TPB) Proliferation in added DMEM.

Example  2: plasmid construction

Standard recombinant DNA technology was used for all plasmid construction. Maps and sequences were obtained from the authors upon request. The original plasmid (pVEEV TC-83) with the VEEV TC-83 genome, under the control of the SP6 RNA polymerase promoter, has been disclosed in several places (33). pVEEV / IRES included EMCV IRES with the first four codons of EMCV polyprotein. This sequence was cloned into the VEEV subgenomic RNA-coding sequence between the end of the 5'UTR and the starting AUG. pVEEV / mutSG / IRES encoded the VEEV TC-83 genome. The subgenomic promoter in the genome was inactivated by clustered point mutations, but this did not modify the amino acid sequence of the carboxy terminal of nsP4 (FIGS. 1A and 1B). This viral genome deleted the 5'UTR of the subgenomic RNA. Thus, VEEV TC-83 nonstructural and structural proteins were expected to be synthesized from the same genomic RNA. Adaptive mutations were induced in pVEEV / mutSG / IRES-encoded nsP2 by PCR amplification of the fragment of interest in the selected variant and replacing the corresponding fragment in the original genome. The same PCR-based technique was used for the synthesis of replicating another fragment from the 3'UTR of the VEEV / mutSG / IRES genome to the Sphl site. All cloned fragments were sequenced before further experiments with the rescued virus.

Example  3: RNA  Warrior

The plasmids were purified by centrifugation to CsCI hardness and linearized by MIuI digestion. RNA was synthesized with SP6 RNA polymerase (Ambion) in the presence of cap analogs (Biolabls, New England). Yield and completeness of transcription were analyzed by gel electrophoresis under non-denaturing conditions. RNA concentration was measured on a FluorChem imager (Alpha Innotech) and the transcription reaction was used for electroporation without further purification.

Example  4: RNA Transfection

Electroporation of BHK-21 cells was performed under the conditions described previously (27). To rescue the virus, 1 mg of in vitro-synthesized viral genomic RNA was electroporated into cells 27, then placed in 100-mm dishes and incubated until cytopathological effects were observed. Virus titers were measured using standard plaque assay in BHK-21 cells (26).

To assess RNA infectivity, 10-fold dilutions of electroporated BHK-21 cells were placed in 6-well Costar plates containing subconfluent, naive cells. 5% CO ₂ After 1 hour incubation at 37 ° C. in the incubator, cells were covered with 2 ml of MEM containing 0.5% Ultra-Pure agarose with 3% FBS. Plaques were incubated at 37 ° C. for 2 days and then stained with crystal violet and infectivity was determined in plaque-forming units (PFU) per mg of transfected RNA.

Example  5: Sequencing of Viral Genomes

Large plaques were randomly selected during titering of viral stock (without staining with neutral red). Virus was extracted from agarose plugs into MEM and a certain amount of the latter medium was used to infect BHK-21 cells in 35-mm dishes. After the development of the profound CPE, virus stocks were harvested for further characterization and RNA was isolated from infected cells by TRIZOL reagent, according to the manufacturer's instructions (Invitrogen). About 1000 nt-long overlapping fragments were synthesized using standard RT-TCR techniques, purified and sequenced by agarose gel electrophoresis. Sequencing of the 5′UTR was performed using the FirstChoice RLM-RACE kit (Ambion) as described elsewhere (16).

Example  6: Viral Replication Analysis

One fifth of the electroporated cells were placed in a 35-mm dish. At the times shown in the figure, the medium was replaced and virus titers measured by plaque analysis in BHK-21 cells. Optionally, BHK-21, NIH3T3 or C ₇ IO cells were placed in a 35-mm dish and infected at the MOI shown in the figure. The medium was replaced with fresh medium and viral titers of harvested samples were determined by plaque analysis in BHK-21 cells.

Example  7: Analysis of Protein Synthesis

The 4 mg of RNA was electroporated into BHK-21 cells and 1/5 of the electroporated cells were placed in 6-well Costa plates. In late 12 h transfection, proteins were metabolically labeled by incubation for 30 min in 0.8 ml DMEM medium lacking methionine and added 0.1% FBS and 20 mCi / ml [ ³⁵ S] methionine. After incubation, they were pooled, transferred to media, pelleted by centrifugation, and dissolved in 100 μl of standard protein loading buffer. Equivalent amounts of protein were loaded onto sodium dodecyl sulfate (SDS) -10% polyacrylamide gel. After electrophoresis, the gel was dried and radiographed.

Example  8: RNA  analysis

To analyze the synthesis of virus-specific RNA, cells were electroporated with 4 mg of in vitro-synthesized viral RNA and one fifth of the cells were transferred to 35-mm dishes. After late 4.5 hours transfection, the medium in the wells was replaced with 1 ml of aMEM with 10% FBS, ActD (1 μg / ml) and [ ³ H] uridine (20 mCi / ml). After 4 hours of incubation at 37 ° C., the whole cellular RNA was isolated by Trizol (Invitrogen) according to the manufacturer's protocol, and then denatured to glyoxal in dimethyl sulfoxide and the conditions described previously And analyzed by agarose gel electrophoresis (7). Gels were immersed in 2,5-diphenyloxazole (PPO), dried and radiographed.

Example  9: IFN Alpha / Beta Analysis

The concentration of IFN-alpha / beta in the medium was measured by the biological assay described previously (41). More specifically, L929 cells were placed in 100 ml of complete medium at a concentration of 5 × 10 ⁴ cells / well in 96-well plates and incubated at 37 ° C. for 6 hours. Medium samples harvested from infected NIH 3T3 cells were treated with UV light for 1 hour and serially diluted in 2-fold steps directly in wells with L929 cells. After 24 hours of incubation at 37 ° C., an additional 100 ml of medium with 2 × 10 ⁵ PFU of vesicular stomatitis virus (VSV) was added to the wells and continued incubation for 36-40 hours. Cells were then stained with crystal violet and the endpoint was determined as the concentration of IFNa / b required to protect 50% of cells obtained from VSV-derived CPE. IFN-a / b criteria for standardization of results were purchased from ATCC and titers of released virus were determined by plaque analysis on BHK-21 cells.

Example  10: Evaluation of Virus Replication in Mosquitoes

In order to assess the replication capacity of mosquitoes in vivo, we used an intra-thoracic inoculation of Aedes aegypti (colonies, Gal., CA) mosquitoes using approximately 10 ⁵ PFU in 1 μl volume. It was. Intrathoracic inoculation has been chosen as an alternative to oral exposure, since almost all of the culicine mosquitoes are very easy to undergo intrathoracic infection by any alphavirus. Oral ease, on the other hand, is highly variable and very less sensitive (42). After inoculation using a glass pipette, the mosquitoes were incubated at 27 ° C. for 10 days and then individually titrated with 1 ml of MEM with 20% FBS and Fungizone. A 100 μl volume of each titrated mosquito was added to Vero cell monolayers on a 24 well plate and observed for 5 days for cytopathological effects to detect infection. Analytical controls included both TC-83 parental virus and IRES mutants.

Example  11: infectious VEEV Immunization and challenge

Six-day-old NIH Swiss mice were inoculated with the VEEV TC-83 strain or designed mutants cerebral (ic) at a dose of approximately 10 ⁶ PFU in a total volume of 20 μl of PBS. After infection, each herd of 8-10 animals was left for 2 months without any manipulation. For 21 days, the mice were observed daily for signs of illness (crumpled hair, depression, anorexia and / or paralysis) and / or death.

Eight week-old female NIH Swiss mice were subcutaneously (sc) vaccinated at approximately 10 ⁶ PFU / mouse using VEEV TC-83 or recombinant virus, followed by about 10 of the highly worsening VEEV strain 3908 after 4 weeks. ⁴ PFUs were challenged subcutaneously. For 21 days, signs of sickness (crumpled hair, depression, anorexia and / or paralysis) and / or death of mice were observed twice daily.

Example  12: recombination VEEV TC -83- Based  virus

The present invention was to develop an alphavirus that can effectively replicate in vertebrate cells that are not mosquito-derived cells. Thus, replication of these viruses had to rely on protein or RNA sequences that function only in vertebrates and not in insect cells. To achieve this, the present invention is designed a method for the expression of alphavirus structural proteins dependent on EMCV IRES. The designed IRES did not contain a poly (C) sequence to obtain the best efficient translation of the VEEV TC-83 structural gene, but retained the first four codons of the EMCV polyprotein. In later experiments, it was confirmed that these additional amino acids had no negative effect on viral replication but had a positive effect on the translation of viral structural proteins (data not shown).

One of the plasmid constructs, VEEV / IRES, replicated the IRES sequence native to the 5'UTR subgenomic RNA downstream (Figure 1A). Thus, it was expected that this genome could synthesize subgenomic RNA. In another recombinant VEEV / mutSG / IRES, the subgenomic promoter was inactivated by 13 very synonymous point mutations (FIGS. 1A and 1B), which was expected to prevent conversion to the active SG RNA promoter. To facilitate the synthesis of VEEV structural proteins, IRES sequences were cloned to replace 26S 5'UTR.

Genomic RNAs of VEEV / IRES, VEEV / mutSG / IRES and unmodified, wild type (wt) VEEV TC-83 were synthesized in vitro and transfected with BHK-21 cells. In infection center analysis, VEEV / IRES RNA showed the same infectivity as the RNA of TC-83 and developed a uniform sized plaque similar to that of TC-83 (FIGS. 1A and 1C). This means that additional adaptive mutations are not required for productive replication of the designed virus. VEEV / IRES replicated with titers in excess of 10 ⁹ PFU / ml, but these final titers and viral replication rates were significantly lower than those of VEEV TC-83 (FIG. 1D). Another recombinant viral genome lacking the subgenomic promoter, BHK-21 cells transfected with VEEV / mutSG / IRES, produced very ineffective infectious virus (FIG. 1D). Pinpoint plaques were developed, and their numbers were difficult to assess. Surprisingly, the virus showed further evolution by serial passage and rapidly developed into variants producing larger plaques (FIG. 1C and data not shown). The growth curve shown in FIG. 1D shows the release of bovine and large plaque-forming viruses.

Thus, the results of these experiments showed that, at least in the context of the VEEV / IRES genome, EMCV IRES can produce structural proteins at levels sufficient for VEEV replication. VEEV / mutSG / IRES, a construct with a mutated subgenomic promoter, produced a defective-in-replication virus that could evolve for more efficient replication.

Example  13: VEEV Of mutSG Of IRES Analysis of adaptive mutations in

The evolution of the VEEV / mutSG / IRES into phenotypes that produce large plaques was presumed to be due to the accumulation of additional mutations in the viral genome. Conversion to the wild type (wt) genomic sequence was not possible due to a number of introduced point mutations and therefore the location of the adaptive mutations was difficult to predict. To identify mutations, randomly select five plaques of VEEV / mutSG / IRES samples harvested in late 24-hour electroporation, and the entire genome of two plaque-purified variants (including 3 'and 5' UTRs) Was sequenced. The list of identified mutations is shown in Figure 2A. Many of these are very synonymous and do not exist in known cis-acting RNA elements. Thus, their effect on virus replication was very unexpected. However, the genome of the two plaque isolates contained the same mutation from the nsP2 protein to Y ₃₇₀ → C, with one of the genomes changing the adjacent encoded amino acid to K ₃₇₁ → Q.

To test the effect of mutations in virus replication, both Y ₃₇₀ → C and Y _37O → C and K ₃₇₁ → Q were cloned into the original VEEV / mutSG / IRES construct (FIG. 2B), RNA infectivity, virus replication rate, plaque The size was compared with that of the original VEEV / mutSG / IRES and other constructs. The same mutations were also cloned into the VEEV TC-83 genome to test their effect on the replication of the parental virus. IRES-encoding genomic RNA with one or both of the mutations in VEEV / mutSG / IRES / 1 and VEEV / mutSG / IRES / 2 showed the same infectivity as the VEEV TC-83 RNA in infection center analysis, and the rescued virus A uniform plaque size similar to that of VEEV TC-83 was formed (data not shown). They showed a strong increase in growth rate (FIGS. 2C and 1D), but the effect of the second mutation was negligible. Thus, the data indicate that the Y ₃₇₀ → C mutation at nsP2 had a strong positive effect on viral replication and the second mutation had no excellent effect. When introduced into the VEEV TC-83 genome, the same mutation did not have a noticeable effect on the replication rate or final titer of the virus (FIG. 2C), which could predict that replication enhancement was specific for the VEEV / mutSG / IRES variant. .

The identified amino acid changes (Y ₃₇₀ → C and K ₃₇₁ → Q) represent only a portion of the possible mutations that can lead to subgenomic promoter-deficiency, effective replication of the IRES-containing virus. Thus, nt 2161-2959 was sequenced in three other plaque clones isolated from the sample via parallel experiments, which were isolated from viral stock after an additional three passages in in vitro-synthesized RNA and Vero cells. Harvest was performed in late 24-hour RNA transfection of 5 plaque-purified variants. This passaging allowed us to select the virus that replicated most effectively. The list of identified mutations is shown in Figure 3A. Sequencing was performed directly from RT-PCR-derived DNA fragments, so the indicated mutations showed consistent sequences in the plaque-derived viral population, but not PCR-derived (FIG. 3B).

All isolates contained mutations in the sequenced fragments, which corresponded to the carboxy terminal fragments of the RNA helicase domain of nsP2. In addition, all altered amino acids were located between amino acids 348-424. In addition, in both the original virus stock and passaged pool generated after electroporation, most common mutations were Y ₃₇₀ → C. This indicates that it probably has one of the most prominent effects in replication, so the above described variant with this particular mutation VEEV / mutSG / IRES / 1 was used in the next experiment.

Example  14: In virus replication nsP2 Y ₃₇₀ → effect of C mutation

The identification of adaptive mutations in carboxy terminal fragments of nsP2-related RNA helicases was surprising and did not give any clear explanation for the increase in VEEV / mutSG / IRES replication. This mutation can enable to have a stimulating effect in RNA replication or viral structural protein translation or viral particle formation or replication complex differentiation and the like. However, the most expected effect was an increase in viral RNA synthesis. Thus, BHK-21 cells were transfected into the synthetic genome in vitro of other VEEV variants, and newly synthesized viral RNA into [ ³ H] uridine in the presence of ActD for 4 hours, starting with late 4.5 hours electroporation. Labeled metabolically, the RNA was analyzed by electrophoresis on agarose gel (FIG. 4A).

As expected, VEEV / IRES can synthesize subgenomic RNA, indicating that the IRES introduced at the 3 'end of the subgenomic RNA 5'UTR did not interfere with subgenomic promoter activity. VEEV / mutSG / IRES and variants thereof with adaptive mutations in nsP2 produced undetectable SG RNA. Thus, 13 mutations introduced into the promoter sequences of these genomes completely destroyed the transcription of the subgenomic RNA. Surprisingly, adaptive mutations in nsP2 are as effective as RNA genome replication in the originally designed VEEV / mutSG / IRES genome, and the cloned VEEV / mutSG / lRES / 1 and VEEV / mutSG / IRES / 2 genomes. It did not have a noticeable effect on RNAs. In addition, genomic RNA replication of all variants was very similar to that of VEEV TC-83. The effects of these mutations were not detected as with the original VEEV TC-83 RNA (see lanes corresponding to VEEV / 1 and VEEV / 2). This finding supported that adaptation did not result in increased RNA replication.

Synthesis of viral structural proteins was assessed in late 12 hours electroporation (FIG. 4B). By that time, VEEV / IRES- and VEEV / mutSG / IRES-specific capsids and similar other structural proteins were synthesized about two times less effective than in cells transfected with VEEV TC-83 RNA. This modest small difference is due to the low infectivity (compared to the titer of VEEV TC-83) of more than 4 and 7 sizes of VEEV / IRES and VEEV / mutSG / IRES viruses, respectively, detected in samples harvested in late 12 h transfection. Does not explain. In addition, differences between viral protein synthesis in BHK-21 cells containing the original VEEV / mutSG / IRES genome versus VEEV / mutSG / l RES / 1 and VEEV / mutSG / IRES / 2 with adaptive mutations in nsP2 are seen. It was not detected in experiments and other experiments. The salient feature of the pattern of labeled proteins in cells infected with IRES-containing viruses was the presence of two additional bands, which were identified by mass spectrometry as the heat-shock proteins Hsp90 and Hsp72. The biological significance of these inductions is not yet clear, but it was speculated that it resulted from some abnormalities of viral structural protein folding leading to stress propagation in cells with structural proteins expressed from IRES.

In further experiments, the intracellular distribution of viral glycoproteins in cells infected with VEEV TC-83, VEEV / mutSG / IRES and VEEV / mutSG / IRES / 1 were evaluated and the presence of these proteins on the cell surface was VEEV-specific antibodies. Analysis by staining with. No noticeable difference in the distribution of glycoproteins was identified. The possibility that adaptive mutations can cause the formation of additional packaging signals in the viral genome has been investigated, and the mutation-containing fragments (corresponding to nt 2533-2950 of the VEEV genome) are cloned into the 3 'UTR of the VEEV / mutSG / IRES genome. Recombinant in vitro synthesized RNA was tested in the infection center assay. No increase in plaque size or virus titer was detected compared to that of the original VEEV / mutSG / IRES.

In another variant, the subgenomic promoter and the VEEV capsid-coding sequence were cloned into the 3'UTR of the VEEV / mutSG / IRES genome to test whether further capsid expression from the subgenomic RNA increased the efficacy of viral replication. This modification also did not have any positive effect on virus titer. Finally, it was analyzed whether VEEV / mutSG / IRES produced subviral particles without genome instead of infectious virus. No such case was observed. That is, cells transfected with VEEV / mutSG / IRES RNA and metabolically labeled with [ ³⁵ S] methionine are subviral that can be detected by ultracentrifugation at a sucrose gradient (data not shown). It was observed that no particles were produced. Thus, the above-described composite assays do not provide a clear mechanistic explanation for the very inefficient replication of the original VEEV / mutSG / IRES or for the positive effects of mutations detected in VEEV nsP2 on replication of IRES-containing viruses. I couldn't. However, the main object of the present invention is the development of VEEV variants, the replication of which depends on the EMCV IRES function, and both VEEV / IRES and VEEV / mutSG / IRES / 1 have been shown to meet this purpose.

Example  15: In mosquito cells and mosquitoes IRES -Dependence VEEV Mutant  a copy

Accumulated data for alphavirus replication clearly shows their genetic instability and high rate of evolution, resulting in the deletion of any non-homologous genes. In particular, these results appear especially when they have a negative effect on viral replication (17, 40). Thus, one of the important issues of the present invention is whether to provide a virus in which the designed EMCV IRES insertion is stabilized and disables replication in mosquito cells. To test this, C ₇ 10 mosquito cells were infected with BHK-21 cells of in vitro-synthesized RNA with VEEV / IRES and VEEV / mutSG / IRES viruses harvested in late 24 hour electroporation. To test whether the entire library of variants released after RNA transfection can replicate in mosquito cells, VEEV / mutSG / IRES instead of VEEV / mutSG / lRES / 1 described above with adaptive mutation Y ₃₇₀ → C in nsP2. Was used.

At the first passage, at 48 hours late infection of C ₇ 10 cells, the titer of VEEV / IRES approached 1.5 × ^{10 10} PFU / ml, and similar titers harvested after the second passage were detected in the stock. (FIGS. 5A and 5B). On the other hand, the titer of VEEV / mutSG / IRES was 150 PFU / ml after the first passage (which seems to reflect the residual virus used for infection rather than the initial virus produced by mosquito cells), and after the second passage after It was below the limit of detection (FIGS. 5A and 5B). In a further experiment, VEEV / mutSG / IRES of plaque-purified variants with adaptive mutations in nsP2 were passaged in C ₇ 10 cells. Infectious virus was not recovered at all after two blind passages.

In another experiment, cliff. Aejip Ti (Ae. Aegypti) were inoculated in the chest mosquitoes to about 10 ⁵ PFU of VEEV TC-83 and VEEV / mutSG / l RES / 1 variant. None of the 17 mosquitoes inoculated with the IRES mutant replicated detectably in A. aggeti, while 17 out of 17 mosquitoes inoculated with the TC-83 parental strain had an average titer of 10 ⁶ PFU / mosquito or higher. All had replicates at detectable levels in CPE analysis. Thus, the IRES-containing VEEV variant VEEV / mutSG / IRES / 1 was unable to replicate in mosquito cells both in vitro and in vivo.

To account for the high titer of VEEV / IRES (which can produce subgenomic RNA variants) after passage in mosquito cells, two individual plaques were randomly selected and IRES-containing fragments of the genome were sequenced. In both isolates, the IRES sequence was no longer present in the viral genome and only 13 and 15 residual nucleotides of the original IRES were found (FIG. 5C). Thus, passage of VEEV / IRES variants in mosquito cells led to the accumulation of IRES-negative variants, and VEEV / mutSG / IRES (lacking a subgenomic promoter) did not develop mutants that could effectively replicate in mosquito cells.

Example  16: VEEV Of mutSG Of IRES /One The variant is Attenuated  Show phenotype

The present invention was directed to the development of VEEV variants that are non-replicable in mosquito-derived cells (and correspondingly in mosquito vectors) but exhibit a more attenuated phenotype in vertebrates than the parental VEEV TC-83. The slower replication rate of the VEEV / mutSG / IRES / 1 variant led to the problem that the virus could not replicate in vertebrate cells with native IFN-a / b production and signaling. However, the present invention did not. The results of the experiments shown in FIG. 6 show that VEEV / mutSG / l RES / 1 replicates with titers above 10 ⁹ PFU / ml in NIH 3T3 cells without defects in IFN-alpha / beta secretion and signaling. Its replication leads to more effective IFN-a / b induction (FIG. 6), but apparently IFN release did not interfere with the already established viral replication. As seen in BHK-21 cells (FIG. 2C), replication of VEEV / mutSG / l RES / 1 was less effective than replication of VEEV TC-83, which is presumed that the IRES-dependent mutants are attenuated in vivo. It became. In addition, after cerebral (ic) inoculation of 6 day old mice at about 10 ⁶ PFU, 86% survived and did not develop signs of encephalitis, whereas 92% of mice received the same dose of VEEV strain TC-83. Died. Taken together, the data showed that the genetically modified IRES-dependent VEEV was more attenuated than the parental VEEV TC-83.

Nevertheless, the VEEV / mutSG / IRES / 1 variant remained immune in both newborn and adult mice. Of the 12 6 day old mice inoculated with VEEV / mutSG / IRES / 1, the wild type VEEV strain 3908 was sc challenged with 10 ⁴ PFU after 5 weeks of 10 administration, whereas 12 Siamese (PBS) Infected mice were challenged in the same manner and none survived (FIG. 7). VEEV / mutSG / l RES / 1 was also immunogenic in adult mice, and sc immunized with about 10 ⁶ PFU after 3 weeks of sc challenge with 10 ⁴ PFU (˜10 ⁴ LD ₅₀ ) of VEEV strain 3908. 80% of mice were protected (FIG. 8). Neutralizing antibody titer (PRNT ₈₀ ) was undetected less than 1:20 in all of these mice immediately prior to challenge, indicating that incomplete protection after a single vaccination resulted in lower IRES-containing virus replication in vivo Backed up. Thus, its high level of attenuation provides a high degree of safety, but repeated vaccinations will probably be required.

Example  17: Keep the lack of mosquito infection Attenuation  Reducing expression strategies

In a separate embodiment of the present invention, a novel expression strategy was designed to maintain lack of mosquito infectivity but reduce attenuation. This strategy involved placing the IRES downstream of the envelope glycoprotein gene at the capsid gene at the 3 ′ end of the subgenomic region immediately before the 3 ′ UTR (FIG. 9). Thus, the envelope protein gene was translated in a cap-dependent manner and the capsid protein was translated in an IRES-dependent manner, forming a subgenomic message. Replication of this new IRES mutant in BHK and Vera cells was effective but was not detected in C ₇ 10 mosquito cells. 20 kids apps Death GT (Aedes aegypti ) Intrathoracic inoculation of adult female mosquitoes did not provide evidence of replication. When this IRES version 2 was used to vaccinate mice, all 10 were seroconverted with an average titer about 2 times lower than that induced by normal TC-83, and all 10 mice were Protected by IRES version 2 from lethal subcutaneous challenge. Thus, the new IRES expression strategy was less attenuated while maintaining the mosquito-competent phenotype.

Immunity and Efficacy of TC-83 IRES Mutants Vaccine Strains Serum Converted Fragments Mean Neutral Ab Titer ± SD Protected Fragments for Deadly VEEV Challenges IRES version 1 1/10 <20 8/10 IRES version 2 10/10 224 ± 260 10/10 TC-83 5/5 576 ± 143 5/5 Sham 0/5 <20 0/5

The following references are cited herein.

Aguilar, PV et al., 2007, J Virol 81: 3866-76.

2. Alevizatos, AC et al., 1967, Am J Trop Med Hyg 16: 762-8.

3. Barton, DJ et al., 1999, J Virol 73: 10104-12.

4. Berge, TO et al., 1961, Am . J. Hyg . 73: 209-218.

5. Blaney, JE, Jr. et al., 2006, Viral Immunol 19: 10-32.

6.Blaney, JE, Jr. et al., 2005, J Virol 79: 5516-28.

7. Bredenbeek, PJ et al., 1993, J. Virol . 67: 6439-6446.

Burke, DS et al., 1977, J Infect Dis 136: 354-9.

9. DaI Canto, M. C, and SG Rabinowitz, 1981, J Neurol Sci 49: 397-418.

10. Davis, N. Letal., 1995, Viroligy 212: 102-110.

11. Davis, NL et al. 1991, Virology 183: 20-31.

12. Davis, N. Letal., 1989, Virology 171: 189-204.

Garmashova, N. et al., 2007. J Virol 81: 13552-65

14. Garmashova, N. et al. 2006,. J Virol 80: 5686-96.

15. Garmashova, N. et al., 2007, J Virol 81: 2472-84.

16. Gorchakov, R. et al., 2004, J Virol 78: 61-75.

17. Gorchakov, R. et al., 2007, Virology 366: 212-25.

18. Griffin, D. E. 2001. Alphaviruses, p. 917-962. In Knipe and Howley (ed.), Fields' Virology, Fourth Edition. Lippincott, Williams and Wilkins, New York.

19. Hart, MK et al., 2000, Vaccine 18: 3067-75.

20. Jan, E., and P. Sarnow. 2002, J MoIBiol 324: 889-902.

21. Johnson, K. and D. Martin. 1974. Adv . Vet . Sci . Comp . Med . 18: 79-116.

22. Khromykh, AA et al., 2001, J Virol 75: 4633-40.

23. Kinney, RM et al. 1993, J. Virol . 67: 1269-1277.

24. Kinney, RM et al. , 1989, Virology 170: 19-30.

25. Kuhn, RJ et al., 1996, J Virol 70: 7900-9.

26. Lemm, JA et al., 1990, J. Virol . 64: 3001-3011.

27. Liljestrom, P. et al., 1991, J. Virol . 65: 4107-4113.

28. Morrill, JC et al., 1991, Vaccine 9: 35-41.

29. Murphy, B. R., and R. M. Chanock. 2001. Immunization against viral diseases, p. 435-467. In D. M. Knipe and P. M. Howley (ed.), Fields' Virology, Fourth Edition. Lippincott, Williams and Wilkins, New York.

30. Nugent, CI et al., 1999, J Virol 73: 427-35.

31. Paessler, S. et al., 2003, J Virol 77: 9278-86.

32. Pedersen, CE et al., 1972, Am J Epidemiol 95: 490-6.

33. Petrakova, O. et al., 2005, J Virol 79: 7597-608.

34. Pittman, PR et al., 1996, Vaccine 14: 337-43.

35.Pugachev , KV et al., 2000, J Virol 74: 10811-5.

36. Rice, CM et al., 1987, J. Virol . 61: 3809-3819.

Rivas, F. et al., 1997, J Infect Dis 175: 828-32.

38. Russo, AT et al., 2006, Structure 14: 1449-58.

39. Strauss, JH, and EG Strauss, 1994., Microbiol . Rev. 58: 491-562.

40. Thomas, J M et al., 2003, J Virol 77: 5598-606.

41. Trgovcich, J. et al., 1996. Virology 224: 73-83.

42. Weaver, S. C. 1997. Vector Biology in Viral Pathogenesis, p. 329-352. In N. Nathanson (ed.), Viral Pathogenesis. Lippincott-Raven, New York.

43. White, L. J. et al., 2001, J Virol 75: 3706-18.

All patents or documents mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. In addition, these patents and documents are incorporated herein by reference in the same amounts if each individual document is indicated to be specifically and individually incorporated by reference.

Claims (18)

Instead of the natural 5'UTR of subgenomic RNA, the interior of the Encephalomyelocardis virus between the end of the nonstructural protein 4 (nsP4) coding sequence and the initiating AUG of the subgenomic RNA coding sequence of the alphavirus. Cloning the ribosomal entry site. The method of claim 1,
Inactivating the subgenomic promoter of the alphavirus by clustered point mutations and deletion of the native 5′UTR in the subgenomic RNA. The method of claim 2,
Inactivation of said subgenomic promoter does not modify the carboxy terminus of non-structural protein 4. The method of claim 2,
And introducing an adaptive mutation into nonstructural protein 2 (nsP2) that is effective for increasing viral replication, release and viral titer. The method of claim 4, wherein
The adaptive mutation is Y ₃₇₀ → O, K ₃₇₁ → Q, P ₃₄₉ → T, D ₄₁ 8 → A, K ₄₂₃ → T or a combination thereof. The method of claim 1,
Said alpha virus is Chikkununya virus. The method of claim 1,
Said alphavirus is Eastern Equine Encephalitis Virus. The method of claim 1,
Said alpha virus is Venezuelan Equine Encephalitis virus. The method of claim 1,
And said alpha virus is Western Equine Encephalitis Virus. An attenuated recombinant alphavirus produced by the method of claim 1. The method of claim 10,
The alphavirus is unable to replicate in mosquitoes, cannot be transmitted by mosquito vectors, high concentrations of IFN alpha / beta, slow growth in IFN alpha / beta cells, induction of slow growth in IFN signaling-deficient BHK-21 cells Or a combination thereof possible. A vector comprising a nucleotide sequence encoding the attenuated recombinant alphavirus of claim 10. A host cell comprising and expressing the vector of claim 12. A pharmaceutical composition comprising the attenuated recombinant alphavirus of claim 10 and a pharmaceutically acceptable carrier. An immunogenic composition comprising the attenuated recombinant alphavirus of claim 10. A method of protecting a subject from infection following exposure to alphavirus, comprising administering an immunologically effective amount of the immunogenic composition of claim 15 to protect the subject from infection following alphavirus exposure. The method of claim 16,
And said object is a human or domestic animal. Cloning the internal ribosomal entry site of the Encephalomyelocarditis virus and the capsid gene downstream of the envelope glycoprotein gene with the capsid gene at the 3 'end of the upstream immediately before the subgenomic region of 3'UTR, wherein the IRES The attenuated recombinant alphavirus, incapable of mosquito infection, characterized in that the capsid is expressed in a cap-independent manner, except for the capsid protein gene translated in a dependent manner, and the envelope protein gene is translated in a cap-dependent manner. Method of creation.

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